Human Decidual NK Cells from Gravid Uteri and NK Cells from Cycling Endometrium are Distinct NK Cell Subsets

dNK and eNK cells present distinct gene expression profiles. A) Unsupervised hierarchical clustering of 4 dNK cell samples and 5 eNK cell samples based on the expression profile of genes with variable expression levels across all samples. 1414 genes were considered in this analysis as they complied with the following criteria: they were expressed by at least 30% of the samples, and σ_i/μ_i ratio was >0.5 and <1000 (where σ_i and μ_i are the standard deviation and mean of the hybridization intensity values of each individual gene across all samples). The resulting dendogram reflects the similarity in gene expression profiles between each of the samples. B) Relative intensity profiles of genes differentially expressed at p < 0.01 with at least 3 fold change in intensity between dNK and eNK cells. Each row represents the relative hybridization intensities of a gene in the different samples. Each column represents one sample. Color intensities reflect the magnitude of relative expression levels of a particular gene across samples. Red represents higher expression, green represents lower expression, and black represents average intensity across samples. C) The transcription profiles of dNK and eNK cells are as different as the transcription profiles of CD56^bright pNK and CD56^dim pNK cells. Number of genes differentially expressed at p < 0.01 between dNK and eNK cells and between CD56^bright, CD56^dim pNK cells and dNK cells. Data correspond to two independent datasets obtained with microarrays HGU133Plus2 for dNK (4 samples) vs eNK cells (5 samples), and with microarrays HGU133A and HGU133B for dNK (4 samples) vs CD56^bright pNK cells (3 samples), dNK (4 samples) vs CD56^dim pNK cells (3 samples), and CD56^bright pNK cells (3 samples) vs. CD56^dim pNK cells (3 samples). The average of genes differentially expressed in comparisons involving all possible combinations of three samples of each cell type is shown to correct for unequal sample sizes. Student's t-test comparisons in this analysis were done with Dchip software. The number of genes differentially expressed normalized by the total number of genes present in each array presented a similar trend (not shown). D) Dendogram of the relationship between the four NK cell subsets based on the data of C.

Genes differentially expressed by dNK and eNK cells. Fold changes of genes that showed greater than or equal to three fold change at P < 0.01 by Student's t-test. 153 genes meeting these criteria were classified into the following categories: membrane proteins/receptors; signal transduction; cytoskeleton related; secreted molecules; metabolism related, DNA binding/transcription/translation; stress related; genes with unknown function; and other genes. Gene, gene name, or gene symbol obtained from Netaffx (Affymetrix, Inc.) GB, GenBank accession number. Probe ID, Affymetrix probeset designation. P-(Presence) calls (dNK, eNK), number of present calls by Affymetrix algorithms in 4 dNK and 5 eNK samples.

Validation of microarray data by RT-Q-PCR. Fold change in the level of transcripts of 10 genes differentially expressed by dNK and eNK cells as evaluated by microarray gene expression profiling (black bars) and RT-Q-PCR (white bars). Microarray data correspond to the average of 5 eNK and 4 dNK samples as described in Fig. 2. RT-Q-PCR values are the average of duplicates obtained from one eNK and one dNK sample and were normalized to the expression level of β-actin, that was similar in both samples (not shown), before calculating the fold change in expression level between the two cell types.