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Transcriptomic profiling by RNA sequencing (RNA-Seq) represents the preferred approach to measure genome-wide gene expression for understanding cellular function, tissue development, disease pathogenesis, as well as to identify potential biomarkers and therapeutic targets. For samples with small cell numbers, multiple methods have been described to increase the efficiency of library preparation and to reduce hands-on time and costs. This chapter reviews our approach, which combines flow cytometry and the most recent high-resolution techniques to perform RNA-Seq for samples with low cell numbers as well as for single-cell samples. Our approach reduces technical variability while increasing sensitivity and efficiency. Thus, it is well-suited for large-scale gene expression profiling studies with limited samples for basic and clinical studies.

Recruitment of Fc-receptor-bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibody's Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor-associated expression of NKG2DL for targeting leukemic cells by NKG2D-immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D-Fc containing a wild-type Fc part (NKG2D-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D-Fc-KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT already enhanced antileukemia reactivity by inducing antibody-dependent cellular cytotoxicity (ADCC) with NKG2D-Fc-ADCC mediating significantly stronger effects. Parallel application of NKG2D-Fc-ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor-associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D-Fc-ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.