Bone Marrow-Derived CD57+ NK and NK-like T-Cells Expand in Lower-Risk MDS and Are Implicated in Driving Bone Marrow Failure
Authors: Rai Richa , Feld Jonathan , Sheppard Richard , Yu Haocheng , Ingles John Cris , Okulate Iyinyeloluwa , Demakos Erin , Hussein Shafinaz , Wiatrowski Kyle , Chen Rachel , Lee Rachel , de Real Ronaldo Miguel , Kelly Geoffrey , Lee Brian , Dawson Travis , Chen Zhihong , Kim-Schulze Seunghee , Silverman Lewis R. , Horowitz Amir 
Published: December 2024

Background: Myelodysplastic syndromes (MDS) are marked by clonally expanded hematopoietic stem cells (HSC), impaired hematopoiesis, and concurrent cytopenia(s). Hematopoietic dysfunction and bone marrow (BM) failure (BMF) are the leading causes of morbidity and mortality in MDS patients (pts). Therapies aimed at restoring hematopoiesis are urgently needed. Approximately 30% of MDS pts present with autoimmune diseases (Fozza et al, 2022). The immune-mediated mechanisms underlying BMF in MDS are not well understood. Impairments in immune cell function have been documented, including abnormalities in natural killer (NK) cells, T cells, regulatory T cells (Treg), and B cells (Tao et al, 2020). Further, it has been established that large granular lymphocyte (LGL) leukemia can co-exist in a BMF spectrum with MDS, Aplastic Anemia, and Paroxysmal Nocturnal Hemoglobinuria (Zhang et al., 2017). Our data confirm that LGLs comprise of both NK and T cells. NK cells, “educated” through interactions between NK cell receptors and HLA class I ligands participate in immune surveillance and target cells with loss of HLA class as “missing self”. Activated NK cells expressing CD16a+ engage autoantibodies targeting specific cells or Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) (Horowitz et al, 2016). We used clinical flow data to establish correlations between CD57+ cells and clinical cytopenias in lower-risk MDS (LR-MDS) to guide deeper exploration of the immune environment in MDS.
Method: A random cohort of 50 MDS pts treated at our institution were studied. We analyzed BM clinical flow cytometry (CFC) data and their banked BM samples taken at different clinical time points. CFC data included characterization of NK cells and T cells using a panel of antibodies comprising CD45, CD3, CD8, CD57, CD56, and CD16/56. We re-analyzed the CFC data using CytobankTM., We categorized each pt based on international prognostic scoring system - mutation (IPSS-M), demographic, diagnostic, laboratory, mutational, and other clinical features of the pts and conducted correlative analyses of these parameters with NK cell and T cell subsets observed by CFC. We performed single-cell RNA sequencing (scRNA-seq), including matched T-cell receptor (TCR) and B-cell receptor (BCR) sequencing, as well as single-nucleotide ATAC seq on BM mononuclear cells (BMMCs) from a separate cohort of six low-risk MDS (LR-MDS) pts and three healthy individuals. We also conducted similar analyses on sorted CD57+ cell populations from three LR-MDS pts and three healthy individuals.
Results: We observed a significantly higher frequency of NK cells in the BM of pts with LR- and Int risk MDS compared to very LR disease (p&lt;0.01) among the cohort. Increased abundance of NK cells was associated with more severe neutropenia (p&lt;0.05) and thrombocytopenia (p&lt;0.001). Transcriptional sc-RNA-seq and CyTOF analyses of BMMCs showed various immune subsets that are expanded or contracted in MDS pts. We profiled the functional states and clonality of CD57+ LGLs, which were uniquely composed of CD8 and gd T cells, and NK cells. The gd T and CD8 T cells expressed a dominant array of inhibitory (e.g. NKG2A, KIR) or activating (e.g. NKG2C, NKG2D) receptors traditionally ascribed to NK cells, affording them “NK innate-like” TCR-independent functions including ADCC and capacity to target cells which have lost class I HLA. In addition to these putative dysregulated immune effector mechanisms, we identified a strong loss of HLA class I in developing hematopoietic cells in timepoint-matched BM from pts with LR/Int Risk MDS. In addition, to dysregulated immune surveillance, MDS pts demonstrated expanded BM NK and innate NK-like CD57+ cells and elevated abundance of autoantibodies in their serum.
Conclusion: Our data uniquely profiles a CD57+(LGL) population in the BM of MDS pts, revealing it to be comprised predominantly of NK cells and NK like T cells. These NK cells may play a pathogenic role in MDS by dysregulating “immune surveillance” mechanisms, via dual mechanism of killing “missing-self” or auto-antibodies targeting HSC and hematopoietic progenitors via “ADCC”. Our observations identify this NK phenotype as a potential target for future therapeutic intervention in MDS that can reverse BMF and improve pt outcomes. Expanded and more detailed data on LGL (NK, gd T and CD8 T cells) function that correlates with HSC targeting will be presented.