In Addition to Immune Suppression, There Is Defective Maturation of CD34+ Cells in Patients with Natural Killer-Lymphoproliferative Disease of Granular Lymphcoytes (NK-LDGL).
Authors: Epling-Burnette P.K. , Bai Fanqi , Painter Jeffrey S. , Loughran Thomas P. 
Published: April 2020

Abstract
Chronic NK-LDGL is characterized by the expansion of CD3−, CD16+ and/or CD56+ mature NK cells associated with anemia and/or neutropenia. There are many reports of concurrent LDGL expansions in patients with other types of bone marrow failure syndromes such as myelodysplastic syndrome (MDS) and aplastic anemia (AA). Bone marrow suppression through autoimmune mechanisms has been suggested to have importance in these diseases along with failed hemaptopoiesis through ineffective stem cell differentiation. The critical mechanism for failed hematopoiesis in patients with NK-LDGL is thought to be through a mechanism of antigen-driven expansion of autoreactive NK cells that delete mature myeloid cells in the peripheral compartments. We found that patients with NK-LDGL express a skewed repertoire of NK receptors. (Epling-Burnette et al, Blood103:3431, 2004) NK cells from these patients expressed a large number of activating NK receptors (NKRs) by genotype analysis and they also had potent cytolytic function in both direct and redirected cytotoxicity assays. Overexpression of activating receptors in the absence of the appropriate inhibitory receptors may cause these cells to recognize and lyse or produce inflammatory cytokines in response to autologous tissues. Therefore, our goal was to examine the colony forming capacity of CD34+- stem cells from patients with NK-LDGL and to determine whether the presence of autologous NK cells influence this maturation process. We isolated peripheral CD34+-stem cells from patients with NK-LDGL and normal controls by fluorescence-activated cell (FACS)-sorting using anti-CD34-PE-conjugated antibodies. Erythroid (BFU-E) and myeloid (CFU-GM) colony formation assays were performed by plating 500 CD34+ cells in methylcellulose in the absence and presence of CD3−/CD56+ NK cells that were simultaneously collected by FACS-sorting. On average, 274 ± 276 erythroid colonies and 253 ± 167 myeloid colonies were obtained from CD34+ cells from normal donors. Compared to each normal donor, CD34+cells from five out of seven NK-LDGL patients showed significantly reduced erythroid colony formation. Five out of six of these NK-LDGL patients also had reduced myeloid colony formation (p≤0.05). Myeloid colony formation was not determined in one patient. In addition to reduced colony formation, two patients demonstrated even further reduction in colony formation, erythroid or myeloid, in the presence of autologous CD3−/CD56+ or CD16+ NK cells. In contrast, there was no difference in colony formation when autologous NK cells were added to normal control CD34+-stem cells. Interestingly, the two patients with NK cell-mediated reduction in colony formation also had reduced functional capacity of their inhibitory NKRs against cross-reactive homozygous MHC-Class-I ligands. These results suggests that deficient levels of inhibitory NKRs were expressed to protect from autologous tissue recognition. These surprising results indicate that patients with NK-LDGL have defective CD34+ hematopoietic stem cells in addition to dysregulated autoimmunity that could contribute to development of failed hematopoiesis. Our findings have critical therapeutic implications for patients with NK-LDGL.