CD56 expression levels on IL-12-modulated <i>ex vivo</i> NK cells.

<p>The intensities of expression of CD56 on cells expressing CD62L, CD16 or KIR was analyzed by flow cytometry analysis on gated CD56⁺<i>ex vivo</i> NK cells differentiated in presence or without IL-12. (A) Dot plots of one representative experiment of four that revealed strong induction levels for CD62L, KIR and CD16 is shown. (B) The statistical analyses of the CD56^bright and CD56^dim distribution for these four independent experiments is given from top to bottom for total CD56⁺, CD62L⁺NKG2A⁺, CD16⁺NKG2A⁺ or KIR⁺NKG2A⁺ cells. Results are displayed as mean percentage ± SEM.</p

Effect of IL12 on CD62L and KIR expression within the developmental subset of CD33⁺NKG2A⁺ cells.

<p><i>Ex vivo</i> NK cells differentiated with or without 0.2 ng/ml IL-12 were analyzed in regard of CD62L and KIR expression within the cell population displaying CD33 and NKG2A expression at day 28 of culture. (A) Flow cytometry dot plots of cells from an IL-12 containing culture are shown to illustrate the gating strategy. By gating on the four quadrants of the dot plot displaying NKG2A and CD33 expressing cells (left panel) the expression of KIR and CD56L on NKG2A⁻/CD33⁻, NKG2A⁻/CD33⁺, NKG2A⁺/CD33⁺ and NKG2A⁺/CD33⁻ cells is depicted (right panel). (B) displays the statistical analyses of the KIR and CD62L expressing cells within the NKG2A⁺CD33⁺ double positive cell population based on 5 independently performed cultures. Results are displayed as mean percentage ± SEM.</p

Regulation of transcription factors important for NK cell differentiation.

<p>Cultures of day 30 were MACS-sorted for CD56 expression. Total RNA was extracted and mRNA levels were analyzed by realtime RT-PCR for nine transcription factors implicated in NK cell differentiation. All values were normalized to β-actin as internal standard. Results are shown as mean values ± SEM calculated from 4 independent experiments performed. To display the results the mean values for the samples differentiated without IL-12 were arbitrarily set to 100%.</p

Effects of different concentrations of IL-12 on the phenotype and the purity of <i>ex vivo</i> generated NK cells.

<p>Effects of high and low doses of IL-12 on the <i>ex vivo</i> NK cell generation and the phenotype of the NK cells were determined by flow cytometry. In a dose-response analysis the effects of concentrations between 10 pg/ml and 20 ng/ml IL-12 on NK cell purity (A, displayed as % CD56⁺ NK cells in the culture) and NK cell receptor expression (B, displayed as % receptor positive cells within the CD56⁺ NK cell subset of the culture) were scored. Expression of CD62L, CD16 and KIR on CD56⁺ cells is depicted. Values are shown as mean ± SD calculated from triplicate wells for one representative experiment at day 28 of culture. (C) An optimal concentration of 0.2 ng/ml IL-12 was chosen for further experiments and analyzed at day 28 of culture. The increase in total cell number (left panel) and the purity of CD56⁺ NK cells (right panel), i.e. the percentage of CD56⁺ cells per total cell counts, was determined. Mean numbers of total cells or percentages of CD56⁺ cells ± SEM for several independent cultures (n) are shown as indicated.</p

Functional assays display a correlation of phenotypical characteristics with improved functions regarding adhesion, cytokine production and cytotoxicity of IL-12-modulated NK cells.

<p>(A) Comparison of adhesion capacity to endothelial cells. <i>Ex vivo</i> generated NK cells from day 28 of cultures including or without IL-12 were purified and subsequently used in adhesion assays on LecTERT lymphatic endothelial cells (LEC) or resting human umbilical vein endothelial cells (HUVEC). Mean values ± SEM calculated from 3 independent experiments each performed in duplicates are shown. (B) Comparison of IFN-γ production capacities. <i>Ex vivo</i> generated NK cells from day 28 were cocultured with target cells at 1∶1 ratio. IFN-γ content was measured in the supernatants by ELISA. Mean values ± SEM calculated from 4 independent experiments are shown. (C) Comparison of NK cytotoxicity. After day 28 of culture <i>ex vivo</i> NK cells differentiated in the presence or absence of IL-12 were tested for their capacity to lyse the MHC class I-deficient target cell line K562 (n = 5) and the MHC class I-positive target cell line KG1a (n = 3). Cytotoxicity was determined on the basis of remaining intact CFSE-labeled target cells and is shown as mean values ± SEM. (D) Comparison of ADCC capacity. <i>Ex vivo</i> NK cells generated with or without 0.2 ng/ml IL-12 resulting in 36% or 21% CD56⁺CD16⁺ cells, respectively, were purified at day 28 of culture and subsequently analyzed in Europium-release killing assays. The human acute lymphoblastic leukemia cell lines 721.221 and Nalm-6 were used at an effector to target ratio of 6∶1 in the presence or absence of rituximab as indicated. Mean values ± SD calculated from triplicate wells are shown for one experiment representative of two performed.</p

Influence of the optimized IL-12 concentration on receptor expression of <i>ex vivo</i> differentiated NK cells.

<p>The effect of 0.2/ml IL-12 on the expression of several NK cell antigens was determined by flow cytometry at day 28 of <i>ex vivo</i> differentiation. (A) Flow cytometry dot plots depicting the expression of CD62L, NKG2A, KIR and CD16 on gated CD56⁺ cells are shown for one representative <i>ex vivo</i> NK cell differentiation culture induced with or cultured without IL-12. (B) Column charts show for several independently performed cultures the IL-12-mediated increase of CD62L, KIR and CD16 positive cells and (C) of CD62L+NKG2A+, KIR+NKG2A+ and KIR+CD16+ double-positive cells. (D) The change of cell numbers with expression of the chemokine receptors CXCR3, CXCR4, CXCR5, CCR6 and CCR7 is shown. The statistical analyses are based on ≥5 independently performed experiments and are displayed as mean percentage ± SEM.</p