In Vitro Generation of NK22 Cells From Hematopoietic Stem Cells
Authors: Tang Qin , Yong-Oon Ahn , Southern Peter , Blazar Bruce R. , Milller Jeffrey S , Verneris Michael 
Published: October 2019

Abstract
Abstract 108
NK cells are the first lymphocytes to recover after allogeneic hematopotiec cell transplantation (allo-HCT). Rapid NK recovery after allo-HCT is associated with reduced treatment related mortality. Because NK cells elaborate inflammatory cytokines (IFN-g) and mediate cytotoxic killing of malignant cells, they are also implicated in graft vs. leukemia reactions. Curiously early after transplant, donor-derived NK cells are hypofunctional and immature. Over the past year, investigators identified a new category of NK cells, called NK22 cells. These cells are present in secondary lymphoid tissue, such as tonsils, lymph nodes and Peyer's patches. Previous investigators have not been able to identify NK22 cells in adult blood or UCB, likely due to lymphoid tissue homing receptor expression (CCR6 and CCR7). NK22 cells are CD56+/−CD117highCD94−IL-1bR+, a phenotype which overlaps with one previously used to describe NK progenitors (i.e., stage III immature NK cells). At present, it is not known whether NK22 cells are a distinct branch of the NK lineage or are NK developmental intermediates. NK22 cells are present in secondary lymphoid tissue at vanishingly small quantities (<1% of all mononuclear cells), thereby making the study of these cells challenging. Functionally, NK22 cells lack of “classical” NK functions (cytotoxicity and IFN-g production) and instead elaborate IL-22 in response to dendritic cell derived IL-1 and/or IL-23. IL-22 does not act on hematopoietic cells, but rather on mucosal tissues to induce proliferation, anti-apoptotic functions and the production of antimicrobial proteins (b defensins). NK22 cells also increase the expression of adhesion molecules on MSCs after co-culture, suggesting a role in secondary lymphoid generation and homeostasis. We have previously used a stromal cell based culture system to study NK development from hematopoietic stem cells. Briefly, CD34+ cells are cultured in the presence of IL-3 (for the first week), FLT-3L, SCF, IL-7 and IL-15 for ~4-5 weeks. At the end of this culture period, functional mature NK cells are obtained. Because this system closely recapitulates ontongeny, we hypothesized that it could be used to study NK22 development. At D28 of culture, we found that 90% (range=88-94%) of cells expressed CD56. Approximately 22% (range=16-28%) had a stage III immature NK cell phenotype (i.e., CD56+CD117highCD94−), of which ~87% (range=77-93%) also expressed IL-bR, a phenotype consistent with NK22 cells (n=5). We next purified CD56− and CD56+ cell populations in these cultures and neither showed IL-22 expression at rest. Following IL-1 and/or IL-23 stimulation, the CD56+ fraction made IL-22 transcripts (by qPCR) and protein by ELISA. We next purified the stage III (CD56+CD117highCD94−) and stage IV (CD56+CD117lowCD94+) fractions and found that only the stage III cells were capable of IL-22 production following IL-1/23 stimulation. Co-culture of NK22 cells (or their supernatant) with MSCs resulted in a >2 log increase in ICAM. Likewise, the supernatant of from activated stage III cells induced keritinocyte proliferation and production of antimicrobial compounds. In vitro derived NK22 cells were compared to freshly isolated NK22 cells from human tonsils and nearly identical staining patterns for ROR-gt, Acyl hydrocarbon receptor, NKp44, NKp46, CD127, CD161, CCR6 and CCR7 were observed. Lastly, purified CD56+CD117highCD94− cells could acquire IL-bR and then further differentiate into stage IV cells (CD56+CD117lowCD94+) in the presence of IL-15. However, this was less likely in the presence of IL-15 and IL-1b, suggesting that NK22 cells are developmental intermediates with specific functions (SLT homeostasis and maintenance of mucosal surfaces and immunity). Depending upon the environmental stimuli, these cells will either maintain their IL-22 producing capacity or develop into cytotoxic lymphocytes. These studies are the first studies to describe the generation of NK22 cells from hematopoietic stem cells. They also allow a better understanding of the developmental requirements and functions of these rare cells. Lastly, this simple culture system creates a new opportunity to use NK22 cells therapeutically to enhance SLT tissue repair and mucosal immunity after allo-HCT.

Disclosures:
No relevant conflicts of interest to declare.