466 Immunophenotypic, Proteomic and Genomic Characterization of Human Cord Blood (CB) Vs Peripheral Blood (PB) CD56+Dim NK Cells: A More Pro NK Phenotype in CB

Track: Contributed Abstracts
Saturday, February 16, 2013, 6:45 PM-7:45 PM
Hall 1 (Salt Palace Convention Center)
*Aradhana Awasthi, PhD , Pediatrics, New York Medical College, Valhalla, NY
*Nancy S Day, PhD , Pediatrics, Columbia University Medical Center, New York, NY
Evan Shereck, MD , Oregon Health and Science University, Portland, OR
Janet Ayello, MS, MT(ASCP) , Pediatrics, New York Medical College, Valhalla, NY
Anthony Sabulski , Pediatrics, New York Medical College, Valhalla, NY
Catherine McGuinn, MD , Pediatrics, Well Cornell Medical College, New York, NY
Carmella van de Ven, MS , Pediatrics, New York Medical College, Valhalla, NY
Megan Lim, MD, PhD , Pathology, University of Michigan, Ann Arbor, MI
Mitchell S. Cairo, MD , Pediatrics, New York Medical College, Valhalla, NY
* Considered equal co-primary first authors

CB is a viable alternative source of allogeneic HSC for the treatment of malignant and non-malignant disease (Cairo et al BBMT 2008, Szabolcs/Cairo et al Sem in Hem 2010).  NK cells play a role in innate and adaptive immunity and are characterized as  CD56+ cell population.  Cytotoxic CD56+dim cells make up 90% of PB NK populations (Shereck/Cairo PBC 2007).  We previously ex-vivo expanded  CB MNC into various phenotypes of CD56+dim and +bright  NK cells (Ayello/Cairo BBMT 2006;Exp. Hematology 2009). NK cell anti-leukemic and anti-rejection activities may be essential to the interplay between GVT effects and GVHD after haploidentical HSCT (Dunbar et al,Haematologica,2008).

Objective: To determine differential expression, immunophenotype and genomic and proteomic signatures in CB vs PB CD56+dimNK cells.

Methods: CB NK CD56 cells (94% enrichment) isolated using a standard kit (Miltenyi Biotec) and sorted into CD3-/CD56bright and CD3-/CD56dim subsets. NKR expression was measured by flow-cytometry. Isolated RNA from CB and PB CD56+dimcells underwent microarray studies (Affymetrix, U133A_2). (Agilent GeneSpring and Ingenuity pathway analyses,IPA). Proteomic performed by LC MS/MS with iTRAQ labeling and analyzed with SEQUEST, ProteinProphet, and INTERACT.

Results: There was no difference in NKR expression of CD16, KIR2DL1, KIR2DS1, KIR2DL2, CD161, NKG2C, NKp44, and NKp46 in CB vs. PB CD56dim. There was a significant difference in CB vs PB CD56+dim NK cells in gene expression including: pro-apoptotic genes: CASP10 (3.1F), TNFSF11 (4.7F), CDC2 (3.0F), BCL2L1 (4.3F), NOTCH2 (1.5F); and cell development: PBX1 (7.6F), IL1RN (5.1F), CD24 (5.3F), CD34 (3.5F), CD55 (2.1F), CCL13 (2.2F). Further, there was a significant change in protein expression, CB vs PB CD56+dimcells over 35 proteins, including CELSR1 (25.0F), BLM (25.0F), BDNF (20.0F), PKD1 (16.7F), NOTCH2 (16.7F), BIRC2 (12.5F), AIFM1 (12.5F), EP400 (5.3F), PBX1 (3.9F), SIRT2 (2.9F), LETM1 (2.9F), and ESR2 (2.4F). qRT-PCR and Western  blot analysis validated the genomic and proteomic results, respectively.

Conclusion: These results suggest that CB vs PB CD56+dim NK cells are more prone to undergo programmed cell death (apoptosis), over expression of numerous pro-apoptotic genes, and may be earlier in development (pro-NK) with  significant over expression of CD34.

See more of: Poster Session 2: Histocompatibility/Alternative Stem Cell Sources
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