Immunophenotypic and Proteomic Characterization of Cord Blood (CB) CD56dim Versus Peripheral Blood (PB) CD56dim NK Cells.
Authors: Shereck Evan , Day Nancy , McGuinn Catherine , Satwani Prakash , van de Ven Carmella , Ayello Janet , Crockett David , Wapner Ronald J. , Elenitoba-Johnson Kojo , Lim Megan , Cairo Mitchell S. 
Published: April 2020

Abstract
Background: Neonates may succumb to serious infection due to deficiencies in adaptive cellular immunity, however, little is known regarding the immaturity of CB NK cell innate immunity (Satwani/Cairo et al Biol Neonate 2005). NK cells are characterized by absent CD3 but expression of CD56dim (90%, cytotoxic) and CD56brigh t (10%, mediator) (Shankaran et al Nature 2001). NK subsets’ function is based on their repertoire of NK receptors (NKRs) (Moretta et al Annu Rev Immunol 2001). Furthermore, the amount of CB NK cells is significantly low in cryopreserved CB for CBT (Cairo et al Transfusion 2005), however, they may play an important role in the graft vs leukemia (GVL) effect post CBT. We recently demonstrated the ability to expand CB into NK phenotype ex-vivo with profound NK cytotoxic activity (Ayello/Cairo et al BBMT 2006).
Objectives: To characterize and compare CB vs. PB NK CD56dim NKR and protein expression.
Methods: We positively selected CB vs. PB CD56+ cells using immunomagnetic beads (Miltenyi) and sorted into CD3−/CD56bright and CD3−/CD56dim subsets. NKR expression was evaluated using CD16, CD158a {KIR2DL1}, CD158a,h {KIR2DL1 and KIR2DS1}, CD158b {KIR2DL2}, CD161, NKG2A, NKG2C, NKG2D, Nkp44, NKp46. Additionally, quantitative proteomic analysis was performed using extracts of CB and PB CD56Dim cells using cleavable ICAT labeling followed by multi-dimensional liquid chromatography and tandem mass spectrometry (MS/MS) (Lim et al Lab Invest 2004).
Results: CB vs. PB CD56dim showed significant increased expression of NKG2A (81.34 ± 1.51 vs. 54.09 ± 4.56, p &lt; 0.03) and NKG2D (94.08 ± 2.36 vs. 77.43 ± 3.93, p &lt; 0.035). There was no significant difference in NKR expression of CD16, KIR2DL1, KIR2DS1, KIR2DL2, CD161, NKG2C, Nkp44, and NKp46 seen in CB vs. PB CD56dim. CB CD56dim vs. CD56bright had significant increased expression of FcgrIII (91.17 ± 1.95 vs. 28.73 ± 11.23, p &lt; 0.03) and KIR2DL2 (31.88 ± 4.39 vs. 3.08 ± 0.99, p &lt; 0.02). Furthermore, there were 33 and 37 proteins over and under expressed by ≥ 2 fold between CB vs. PB CD56dim NK cells, respectively. CB CD56dim overexpressed functional proteins were 46% binding, 17% catalytic, 15% signaling, 15% transcription, 3% enzyme, 2% structural, and 2% transport. CB CD56dim underexpressed Inositol 1,4,5-triphosphate receptor type 3, mitogen-activated protein kinase 5, and natural cytotoxicity triggering receptor 3 precursor by 8.33, 6.67, and 2.5 fold decrease, respectively. CB CD56dim overexpressed neurogenic locus notch homolog protein 2 precursor, pleckstrin homology domain-containing family A member 1, and transcriptional repressor NF-X1 by 16.67, 7.14, and 5.26 fold increase, respectively.
Conclusion: While there are many similiarities in CB vs. PB CD56dim, CB CD56dim appear to be quite mature in development compared to other CB immune cell subsets and in fact have overexpression of a significant number of proteins including NKG2D, NKG2A, and several (33) important functional proteins. These studies suggest that CB CD56dim NK cells are similar in development to PB CD56dim NK cells and may contribute to the GVL effect post UCBT.