Introduction: We have previously reported that the source of the graft for allogeneic hematopoietic stem cell transplantation (bone marrow (BM) vs. G-CSF mobilized peripheral blood (G-PB)), and the content of plasmacytoid dendritic cells (pDCs) in the graft are factors associated with improved long-term clinical outcomes. We have also shown differences in homing receptor expression on donor pDCs from BM vs. G-PB grafts that suggest important functional differences related to GvHD and rejection outcomes. Here, we have extended our analysis to study differences in gene expression in pDC and CD34+ hematopoietic progenitor cells from graft types including BM, G-PB, umbilical cord blood (CB), plerixafor-mobilized PB (PLX-PB) and FLT3L-mobilized PB (FLT3L-PB). We hypothesized that comparisons of expression array data from the various graft types would reveal additional differentially-expressed genes and pathways that may have importance in allo-HSCT outcomes, and suggest strategies for improving allo-HSCT procedures through graft selection or screening.

Methods: Aliquots of donor hematopoietic stem cell transplant (HSCT) grafts were obtained under IRB-approved clinical research protocols. These graft types included BM (N=4), CB (N=4), G-PB (N=4), PLX-PB (N=3), and FLT3L-PB (N=5). CD45+CD34+ hematopoietic progenitor cells and CD45+BDCA4+ pDCs were sorted into separate tubes using a FACS Aria cell sorter. The purified cell populations were pelleted and processed for RNA extraction. cDNA was amplified and labeled using a NuGen Ovation Pico system, and gene expression data was generated using the Illumina Human HT-12_V4 array at the Emory Integrated Genomics Core. Raw microarray data were preprocessed, quantile normalized, background-corrected and log 2 transformed prior to differential gene expression analyses using the Limma package to make comparisons between groups, using volcano plots and heatmaps to visualize the data. Pathway analyses were then performed on the differentially expressed genes.

Results: Selecting for pathways that showed differential expression in multiple comparisons, we identified the defensins and alpha-defensins pathways as representing a gene family of interest that varies significantly for both pDCs and CD34+ cells from different graft sources. Defensins had significantly higher expression (p < 0.05) in pDCs from BM, G-PB and CB compared to PLX-PB, and in BM and G-PB compared to FLT3L-PB. The average fold-differences in defensin family gene expression levels were calculated, with pDCs derived from PLX-mobilized grafts having the lowest expression. Defensin gene expression in PLX-PB pDCs was arbitrarily set to 1 as a reference value, and the fold-increase was calculated for pDCs from FLT3L-PB (2.5), CB (33), BM (52) and G-PB (104) grafts. Similar results were found for hierarchical expression of defensin genes in sorted CD34+ cells from the 5 graft types.

Conclusions: Different mobilization strategies result in altered levels of defensin family gene expression in purified pDC and CD34+ hematopoietic progenitor cells. Enhanced levels of defensin expression in mature pDC and myeloid progeny of CD34+ progenitor cells are predicted to improve resistance to opportunistic infections. Classification of defensin levels in these graft sources may be prognostic in identifying transplant patients with an increased risk of infection.


Waller: Coulter Foundation: Research Funding; PRA: Consultancy; AMGEN: Consultancy; Chimerix: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Celldex: Consultancy; Helocyte: Consultancy; Katz Foundation: Research Funding; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Cerus: Equity Ownership; National Institutes of Health: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.