Immunophenotypic and Proteomic Characterization of Cord Blood (CB) CD56^dim Versus Peripheral Blood (PB) CD56^dim NK Cells.

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 CD56^dim (90%, cytotoxic) and CD56^{brigh 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 CD56^dim NKR and protein expression.

Methods: We positively selected CB vs. PB CD56⁺ cells using immunomagnetic beads (Miltenyi) and sorted into CD3⁻/CD56^bright and CD3⁻/CD56^dim 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 CD56^Dim 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 CD56^dim showed significant increased expression of NKG2A (81.34 ± 1.51 vs. 54.09 ± 4.56, p < 0.03) and NKG2D (94.08 ± 2.36 vs. 77.43 ± 3.93, p < 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 CD56^dim. CB CD56^dim vs. CD56^bright had significant increased expression of FcgrIII (91.17 ± 1.95 vs. 28.73 ± 11.23, p < 0.03) and KIR2DL2 (31.88 ± 4.39 vs. 3.08 ± 0.99, p < 0.02). Furthermore, there were 33 and 37 proteins over and under expressed by ≥ 2 fold between CB vs. PB CD56^dim NK cells, respectively. CB CD56^dim overexpressed functional proteins were 46% binding, 17% catalytic, 15% signaling, 15% transcription, 3% enzyme, 2% structural, and 2% transport. CB CD56^dim 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 CD56^dim 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 CD56^dim, CB CD56^dim 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 CD56^dim NK cells are similar in development to PB CD56^dim NK cells and may contribute to the GVL effect post UCBT.