Peripheral Blood (PB) CD56bright and CD56dim Do Not Exhibit Differences in NKR Expression Compared to Their Cord Blood (CB) Counterparts: CB CD56dim May Represent an Intermediary between CB CD56bright and PB CD56dim.

Abstract NK cells are characterized by absent CD3 and expression of CD56. CD56 NK cells are classified into CD56dim (90%) that are primarily cytotoxic, and CD56bright that secrete cytokines (Kaplan et al PNAS 1998; Shankaran et al Nature 2001). NK subsets carry out their respective functions based on their repertoire of NK receptors (NKRs), both activating and inhibiting (Moretta et al Annu Rev Immunol 2001). PB CD56dim cells express high concentrations of killer-cell immunoglobulin receptors (KIR) and C-type lectin receptors. PB CD56bright NK cells have increased expression of C-type lectin receptors, but have low expression of the KIRs (Nagler J. Immunol 1989). CD16 (the Fcγ receptor III or FcγRIII) which is responsible for binding to antibody-coated targets and initiating antibody-dependent cellular cytotoxicity (ADCC) has increased expression in CD56dim vs CD56bright NK cells (Leibson et al Immunity 1997). While the characterization and function of PB NK subsets and NKR expression are well described, there is little information regarding CB NK subsets and NKRs. We previously demonstrated a significant increase in CB NK NKR expression in ex-vivo engineered CB MNC following 48 hours of induction with IL-2, IL-7, IL-12, and Anti-CD3 (Ayello/Cairo et al BBMT 2006). In this study, we compared PB vs. CB NK subsets and NKR expression. PB and CB cells were positively selected for CD56+ using CD56 magnetic beads (Miltenyi, Aubrun, CA). CD56+ cells were sorted into CD3−/CD56bright and CD3−/CD56dim subsets by FACS sorter and NK receptor expression (CD16, CD158a {KIR2DL1} CD158a, h {KIR2DL1 and KIR2DS1}, CD158b {KIR2DL2}, CD161, NKG2A, NKG2C, NKG2D, Nkp44, NKp46, Becton Dickinson, Mountainview, CA,) of each subset was analyzed by flow cytometry. CD56+ selection yielded >89% purity (PB-96%, CB-89%). There was no statistical difference (mean ± SEM) in the receptor expression between the CB CD56bright and PB CD56bright and also between the CB CD56dim and PB CD56dim. However, PB CD56bright vs. PB CD56dim had increased expression of NKG2A (93.28 ± 2.83 vs. 66.64 ±7.74, p< 0.015), NKG2C (62.11 ±11.79 vs. 8.08 ± 2.98, p< 0.12), NKp44 (61.52 ±10.35 vs. 3.79 ± 2.32, p< 0.005) and NKp46 (92.53 ±3.31 vs. 64.66 ± 12.34, p< 0.05). PB CD56dim had increased expression of CD16 (93.30 ± 2.71 vs. 61.15 ± 11.79, p< 0.07) compared to PB CD56bright. The CB CD56dim compared to CB CD56bright has increased CD16 expression (85.06 ± 6.75 vs. 40.91 ± 5.74, p< 0.004) only. The PB CD56bright only differed from the CB CD56dim with respect to NKp44 (p< 0.021). The CB CD56bright differed from the PB CD56dim with respect to KIR2DL1 (p< 0.026), CD161 (p < 0.05), NKG2A (p < 0.025), and NKG2C (p < 0.05). In conclusion, there does appear to be a definite difference in NKR expression in PB CD56bright vs. PB CD56dim. However, PB vs. CB CD56dim and PB vs. CB CD56bright did not have any statistically significant NKR expression differences suggesting that the subsets have similar phenotypes in their respective cell source. Based on these results, we postulate that the CB CD56dim subset might reflect an intermediary step in development transitioning from CB CD56bright to PB CD56dim.